KR100813107B1 - Whitening Composition Containing Fermentation Product of Galla Rhois By Lactobacillus - Google Patents

Abstract

The present invention relates to a whitening composition comprising a gall bladder fermented by lactic acid bacteria, a gall bladder lactic acid bacteria fermented with increased tyrosinase inhibitory activity and a method for preparing the same, and a whitening cosmetic composition comprising the gall bladder lactic acid fermented product and its It relates to a manufacturing method.

Gall bladder, lactic acid bacteria, fermented product

Description

Whitening Composition Containing Fermentation Products by Lactic Acid Bacteria {Whitening Composition Containing Fermentation Product of Galla Rhois By Lactobacillus}

1 shows the invasion of melanin pigment into the skin. The skin is divided into the epidermis, the dermis and the subcutaneous layer from the outside to the inside. Melanin is formed by melanocytes in the dermis, secreted and transported to the epidermis. After moving to the epidermis, melanin pigmentation occurs, causing blemishes, freckles, and browning of the skin.

2 shows the production of melanin from tyrosine. Dihydroxyphenylalanine (L-DOPA) is produced by the hydroxylation reaction of tyrosinase from tyrosine, and dopaquinone is produced through oxidation by the same enzyme. This dopaquinone is finally polymerized to produce melanin. Melanin is produced in two types. One is phenomelanine and the other is eumelanine. Tyrosinase also plays a role in the production of eumelanin. The typical action of whitening cosmetics is to inhibit the action of tyrosinase. Therefore, inhibition of tyrosinase inhibits the production of melanin because L-DOPA, dopaquinone and eumelanin precursor are not produced. The red X marks the site of tyrosinase inhibition.

Figure 3 shows that the lactobacillus fermentation of the gall bladder shows higher tyrosinase inhibitory activity than the gall bladder. AB stands for arbutin, GR stands for Galla Rhois ( Fall ), and FGR stands for fermented Galla Rhois. Fermented five-fold fermented product refers to the fermented lactic acid bacteria fermented Lactobacillus Casei PM1 in the medium containing the five. Arbutin and gall bladder were diluted in tenths of 10 mg per ml of each solid state (in this case, the final dose was 1 mg / ml) or in dilutions of five (in this case the final dose was 2 mg / ml). In the case of fermented Lactobacillus Lactobacillus, 2% (20g) of finely divided liver magella was added to 1 L of lactic acid bacterium medium and fermented Lactobacillus Lactobacillus contained 20 mg of Glucose per ml. Lactobacillus fermentation was administered so that the total dose of gall bladder was 2 mg / ml, and when 50 μl, the total dose was 1 mg / ml.

The color of skin and hair of humans and mammals is determined by several factors, but among them, melanin pigment synthesized in melanocytes is known to play the most important role (see FIG. 1). There are two types of melanin pigments synthesized in melanocytes: eumelanin, which is brown and black, and pheomelanin, which is yellow or reddish brown, and these pigments dopa tyrosine in melanosomes. It is formed by the action of tyrosinase to convert to and dopaquinone (see Fig. 2). Eumelanin is a homopolymer of 5,6-dihyroxyl indole produced by redox of dopaquinone and piomelanin is derived from cysteinyldopa, which is dopaquinone. It is known that the difference in skin color according to race is determined by the difference in the degree and the synthetic ability of these melanin pigments (Burnett, JB, Seiler, H., Brown, JC Separation and characterization of multiple forms of tyrosinase from mouse melanoma. 27: 880-889, 1967; Hearing, VJ, Nicholson, JM, Montague, PM, et al. Mammalian tyrosinase: Structural and functional interrelationship of isozyme. ., Kanoh, H., Jimbow, K. Characterization of biochemical properties of melanosomes for structual and functional differentiation: Analysis of the compositions of lipids and proteins in melanosomes and their subfractions.J. Invest.Dermatol. 79: 97-102, 1982. 6.Prota, G. Recent advances in the chemistry of melanogenesis in mammals.J. Invest.Dermal. 75: 122-126, 1980 .; Sober, AJ Fitzpatrick, TB Pathophysiology of melanin pigmentation in man.Prog Chem Org Natural Products. 31: 521-582, 1974.).

The inventors of the present invention found that the tyrosinase inhibitory activity was increased to enhance the tyrosinase inhibitory activity of the lactic acid bacteria fermentation of the gall bladder, and thus can be used as a raw material for whitening cosmetics. It was.

An object of the present invention relates to a gall bladder lactic acid bacterium fermented product composition with increased tyrosinase activity, a method for preparing the same, and a whitening cosmetic including the gall bladder lactic acid fermented product and a method for producing the same.

In order to achieve the above object, the present invention provides a method for producing a fermentation product with increased tyrosinase inhibitory activity, characterized in that the fermentation is used as one of the fermentation substrates, inoculated with lactic acid bacteria. More preferably, the lactic acid bacteria is Lactobacillus casei ( Lactobacillus casei ) provides a method for producing a fermentation product with increased tyrosinase inhibitory activity, characterized in that. Lactobacillus in the specification of the present invention refers to a bacterium producing lactic acid as a final metabolite by fermenting carbohydrates, lactic acid bacteria are commonly used in the manufacturing process of fermented foods such as kimchi or yogurt. The lactic acid bacteria to be used in food products and the like Enterococcus (Enterococcus) genus Lactobacillus bacteria (Lactobacillus) genus Lactococcus (Lactococcus), A flow Pocono stock (Leuconostoc), A Streptococcus (Streptococcus) in, Wei Cell rasok. The lactic acid bacteria that can be used in the method of the present invention may be used by mixing one or more of various lactic acid bacteria such as bifidus bacteria, Lactobacillus bacteria and Lactococcus bacteria, and among them, it is preferable to use Lactobacillus bacteria. Even more preferably, the lactic acid bacteria is Lactobacillus casei PM1 (KCCM 10766P).

A second aspect of the present invention provides a fermentation composition with increased tyrosinase inhibitory activity made according to the production method of the gall bladder lactic acid bacteria fermentation.

According to a third aspect of the present invention, there is provided a whitening cosmetic comprising the above gall bladder lactic acid bacteria fermentation composition. The whitening cosmetic composition of the present invention is used for the purpose of skin whitening, there is no particular limitation in the formulation, for example, softening water, nourishing cosmetics, massage cream, nutrition cream, pack, gel or skin adhesive type It may be a cosmetic composition having a formulation of a cosmetic, and may also be a transdermal dosage form such as a lotion, ointment, gel, cream, patch or spray. In addition, in each formulation, other components can be appropriately selected and blended by those skilled in the art without difficulty depending on the kind or purpose of use of other cosmetic compositions. In the present invention, in order to obtain the desired effect, the above five gall lactic acid bacteria fermentation is contained in an amount of 0.01 to 10% by weight based on the total weight of the composition. When used in less than 0.01% by weight it is difficult to expect the proper efficacy, when used in excess of 10% by weight may adversely affect the safety and stability of the product.

A fourth aspect of the present invention provides a functional food comprising a lactobacillus fermentation product of the gall bladder. The reason why lactic acid bacteria are of particular interest is 'generally regarded as safe' microorganisms, which are widely regarded as safe because they have been widely used and consumed in foods in both East and West. Lactic acid bacteria fermentation product of gallnuts produced using these safe organisms can be stably used as food. The food containing the gall bladder lactic acid bacteria fermentation may be food such as juice, yogurt, cheese, probiotic. In addition, the gall bladder lactic acid bacteria fermented product can be used as a food additive. The food additive may be added to the general food, so that the general food has a whitening effect.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are only for illustrating the present invention, and the contents of the present invention are not limited by the following examples.

EXAMPLES Preparation of Mellitus Fermented Products

Fermentation condition

Galla Rhois was purchased from Omniherb Pharmaceutical company (seoul, Korea). L-DOPA and mushroom tyrosinase were purchased from Sigma Chemical Co., USA. The gall bladder was ground and fermented with Lactobacillus casei PM1 (KCCM 10766P). In the case of gallnut extract, 100 g of gallnut was ground in a grinder, put in 1 L of distilled water, cut off at 100 ° C. for 1 hour, filtered, and the extract was obtained by lyophilization.

Fermentation of gall bladder by Lactobacillus

Lactobacillus casei PM1; fermentation Gallic by (Lactobacillus casei PM1 KCCM 10766P) was conducted as follows. Inoculate KCCM10766P (preserved in MRS agar) in MRS medium (Difco, 100 ml) having the following composition and incubate overnight (incubation time: 24hr-30hr, when OD value is 0.6 or more at 610 nm) to 100 ml. Inoculum was prepared. Inoculum (100 ml) was inoculated in 900 ml of medium containing 2% (20 g) gall bladder and 1% (10 g) glucose. In order to incubate 1 L, a German fermenter, B. Braun Biotech International, was used as a model BIOSTAT B fermenter. The initial pH was adjusted to 6.5 and automatically adjusted to 6.5 by 1M NaOH solution for 48 hours of incubation time. Samples were measured for anti tyrosinase activity using L-DOPA substrate.

MRS medium (component ratio per liter)

Proteos Peptide Number 310.0g

Beef Extract10.0g

East Extract 5.0g

Dextrose 20.0g

Polysorbate 80 1.0g

Ammonium Citrate 2.0g

Sodium acetate 5.0g

Magnesium Sulfate 0.1g

0.05 g manganese sulfate

Dipotassium phosphate 20.g

EXAMPLES Measurement of Tyrosinase Inhibitory Activity

Measurement of Tyrosinase Inhibitory Activity

Tyrosinase activity was used by slightly modifying the method of Pomerants SH et al. (Pomerants SH, 1963 J. Biol. Chem., 238, 2351-2357). 40 μl of 5 mM L-DOPA solution, 80 μl of 67 mM phosphate buffer solution (pH 6.8) and 40 μl (gallon, arbutin test solution concentration 10 mg / ml) with or without test sample solution were mixed. Next, 40 µl (100 units / ml) of mushroom tyrosinase was added to the reaction mixture. At this time, the final concentration of the test solution was 2mg / ml. To ensure the final concentration of the test solution was 1 mg / ml, 40 μl of 5mM L-DOPA solution, 100 μl of 67mM phosphate buffer solution (pH 6.8) and 20 μl of the concentration of 10 mg / ml of arbutin Those with or without test sample solutions were mixed and then 40 μl (100 units / ml) of mushroom tyrosinase was added to the reaction mixture and tested. Lactobacillus fermentation solution of gall bladder, with or without 40 μl 5mM L-DOPA solution, 100 μl 67mM phosphate buffer solution (pH 6.8) and 20 μl (5 g Lactobacillus fermentation solution concentration 20 mg / ml) What was not mixed. Next, 40 µl (100 units / ml) of mushroom tyrosinase was added to the reaction mixture. At this time, the final concentration of the lactic acid bacteria fermentation solution of gall bladder was 2mg / ml. For the final concentration of lactic acid bacterium fermentation solution of 5 folds, 40 μl of 5mM L-DOPA solution, 110 μl of 67mM phosphate buffer solution (pH 6.8) and 10 μl (lactic acid bacteria fermentation solution concentration 20 mg / ml) Those with or without test sample solutions were mixed and then 40 μl (100 units / ml) of mushroom tyrosinase was added to the reaction mixture and tested. Due to the formation of dopachrome (ε ₄₉₀ = 3600 M ⁻¹ · Cm ⁻¹ ), the increase in absorbance at 490 nm was obtained as a function of time using spectrophotometers (Molecular Devices, Precision Michaelo Plate, USA). Was monitored. Initial rates were used to measure tyrosinase activity. The percentage inhibition of tyrosinase activity was calculated as follows.

% Suppression = [(A-B) / A] x100

Where A is the absorbance at 490 nm without a sample and B is the absorbance at 490 nm when the sample is present.

In order to check the tyrosinase inhibitory activity of the fermentation, arbutin and unfermented gallnuts were tested as controls. Arbutin and gall bladder were diluted in tenths of 10 mg per ml of each solid state (in this case, the final dose was 1 mg / ml) or in dilutions of five (in this case the final dose was 2 mg / ml). In the case of fermented Lactobacillus Lactobacillus, 2% (20g) of finely divided liver magella was added to 1 L of lactic acid bacterium medium and fermented Lactobacillus Lactobacillus contained 20 mg of Glucose per ml. Lactobacillus fermentation was administered so that the total dose of gall bladder was 2 mg / ml, and when 50 µl was administered, the total dose was 1 mg / ml.

result

As shown in FIG. 3 , lactobacillus fermentation of the gall bladder shows higher tyrosinase inhibitory activity than the gall bladder. AB stands for arbutin, GR stands for Galla Rhois ( Fall ), and FGR stands for fermented Galla Rhois. Fermented gall bladder culture means the lactic acid bacteria fermented product fermented Lactobacillus Casei PM1 in the medium containing the gall bladder .

Example 3 Composition of Mellitus Fermented Product

The composition of the five-fermented fermented product was examined through LC-Mass component analysis.

Liquid chromatography-mass spectrum analysis method

To quantitatively analyze glycolic acid, azelaic acid, nicotinamide, L-lactic acid, kojic acid, 4- (benyloxy) phenol, and vitamin C from the gall bladder and gall bladder fermentation, the gall bladder and gall bladder fermentation were added to 10 ml of distilled water. Thawed and filtered through a 0.45 μM dice filter (PVDF Acrodisk ELC, Waters, USA) and the standard was Capsule Pack C18, 2.0 × 50 mm, 5 μm; Elution conditions of ACN (2%) / 0.1% water, formic acid (98%) 200 μl / min; Multi SIM mode negative and column temperature were analyzed at 30 ° C.

Composition of Mungja Lactic Acid Bacteria Fermentation

As shown in Table 1, the fermented lactic acid bacteria fermented glycolic acid (Glycolic acid), azelaic acid (Azelaic acid), nicotinamide, l-lactic acid, 4- (benzyloxy) phenol (4- (benzyloxy) phenol), which is richer in vitamin C.

[Table 1] Composition comparison table of five gall extract and five gall lactic acid fermentation products

Standard material  gallnut Fermented Lactic Acid Bacteria Glycolic acid 0.231 μg 8.85 μg Azelaic acid ND 40.9 μg Nicotinamide 0.227 μg 9.55 μg Lactic acid 2.359 mg 92.45 mg 4- (benzyloxy) phenol 2.51 μg 120.6 μg Vitamin c 0.126 μg 6.3 μg

Glycolic acid, nicotinamide, lactic acid, 4- (benzyloxy) phenol, and vitamin C, which are known to exhibit tyrosinase inhibition and whitening effects, are 38, 42, 39, 48, and 50, respectively, compared to natural gall bladder. It showed a high content of pears. Especially in the case of lactic acid, the content was very high in mg unit. On the other hand, azelaic acid, which was not detected in natural gall bladder, was detected in gall lactic acid bacteria fermentation. The known effects of each of the components found to be increased in gall bladder lactic acid bacteria fermentation are as follows. Lactic acid is known to have whitening, exfoliation, moisture increase effect, wetting, moisturizing, skin pH maintenance and antibacterial effect. Nicotinamide is known for its whitening, moisturizing and anti-wrinkle effects. Vitamin C is known to inhibit tyrosinase, inhibit melanin production, and remove fine wrinkles. Glycolic acid is known to inhibit tyrosinase, inhibit the production of melanin pigment, exfoliate, and block UV rays. It is known that azelaic acid can exfoliate melanin quickly by removing keratin, thereby obtaining a whitening effect. 4- (benzyloxy) phenol is known to inhibit tyrosinase activity and inhibit the production of melanin pigment. Therefore, it is judged that the tyrosinase inhibitory effect was increased as shown in FIG. 3 by these components or the unknown component.

Example 4 Preparation of Mellitus Lactic Acid Bacteria Fermented Product

First, using the gall as one of the substrates, the fermented product produced by fermentation using lactic acid bacteria was centrifuged at 3,000 rpm for 30 minutes to 1 hour to obtain a supernatant, and then, the Whatman filter paper 2 was used to remove the lactic acid bacteria. . In order to remove the lactic acid bacteria more completely, almost all the lactic acid bacteria were removed once again using a 0.45 μM micro filter. It was stored frozen at -20 ° C or in a deep freezer at -70 ° C. When used after storage, dissolved at room temperature and used.

Fermented Lactic Acid Bacteria Fermented by lactic acid bacteria according to the present invention showed a superior tyrosinase inhibitory effect by 16-18% or more than when directly using conventional natural gall bladder, and about 21% than arbutin known as a typical representative whitening agent. It showed high anti-tyrosinase inhibitory effect. Therefore, the fermented fermented product by lactic acid bacteria is expected to have a more excellent melatonin production inhibitory effect, the composition comprising the same can be used as an excellent whitening agent or whitening cosmetics.

Claims (7)

A method for producing a fermented product with increased tyrosinase inhibitory activity, wherein the fermented gall is used as one of the fermentation substrates and inoculated with lactic acid bacteria. The method of claim 1, wherein the lactic acid bacteria Lactobacillus casei ( Lactobacillus casei ) fermentation method with increased tyrosinase inhibitory activity, characterized in that. The method of claim 1, wherein the lactobacillus Lactobacillus casei PM1 ( Lactobacillus casei PM1; KCCM 10766P) characterized in that the fermentation method with increased tyrosinase inhibitory activity, characterized in that. Fermented product with increased tyrosinase inhibitory activity made according to any one of claims 1 to 3. A whitening cosmetic comprising the fermentation product of claim 4. Food comprising the fermentation product of claim 4.       The food of claim 6, wherein the food is selected from the group consisting of juice, yogurt, cheese.

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